It is widely recognized that the human body's response to injury is complex, and is dependent on a panoply of signaling pathways expressed by several cell and tissue types over an extended period of time. Importantly, the wound “bed” undergoes significant remodeling as re-epithelialization ensues concomitant with dermal angiogenesis. And, while wound healing typically occurs as a natural, uneventful process leaving the individual with neither noticeable scars nor wounds that chronically persist, this is not the case for 2-3% of the U.S. population. For these individuals, excessive scarring and chronic wounds are sustained as medical issues requiring specialized treatment, individualized care, or in some cases, hospitalization. Thus, while acute wound healing may occur in a matter of days or weeks, chronic wounds can remain in an open state for months and even years.
Under normal circumstances, the process of human wound healing can be broken down into three phases. An initial inflammatory phase, which is followed by robust tissue remodeling and proliferation (the proliferative phase), is ultimately succeeded by a “maturational phase” in which reepithelialization, dermal angiogenesis, and wound closure ensue. The inflammatory phase is characterized by homeostasis, with a provisional matrix contributed by the blood itself creating the initial wound bed. As basement membrane and interstitial collagens are exposed during injury, blood platelets are stimulated to release multiple chemokines, including epidermal growth factor (EGF), fibronectin, fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin, and von Willebrand factor, to name several. These factors help to stabilize the wound through clot formation and control bleeding, therein limiting the extent of injury. Platelet degranulation also initiates the complement cascade, specifically via C5a, which is a potent chemoattractant for neutrophils.
The timeline for cell migration in a normal wound-healing process is also well ordered, with an inflammatory phase beckoning the migration of immune response cells. For example, neutrophils function to decontaminate the wound from foreign debris via phagocytosis with support from immigrating macrophages. In turn, macrophages release cytokines that locally function to stimulate a robust proliferative response required for tissue morphogenesis and healing. It is during the proliferative phase that re-epithelialization and angiogenesis predominate. The entire process represents a dynamic and reciprocal continuum, with the angiogenesis of wound healing propagating and sustaining the re-epithelialization and tissue remodeling processes: all events continued until the wound site reaches maximal strength, perhaps as long as 1 year post-injury.
Throughout life, the vasculature undergoes significant morphogenesis. Two independent but related processes govern the formation of the adult vasculature: vasculogenesis and angiogenesis. Initially during vasculogenesis, immature vessels are formed de novo from endothelial cell precursors, the angioblasts, which proliferate and coalesce, creating a capillary plexus. Local differentiation of endothelial cells serves as an initiating event for the subsequent rounds of vascular “budding” or “sprouting,” angiogenesis, which gives rise to the system of arteries, veins, arterioles, venules, and capillaries. Interestingly, in the adult, physiologic angiogenesis occurs during the female reproductive cycle, but otherwise the pre-dominant form of physiologic angiogenesis during adult life occurs during wound healing.
Many positively and negatively acting factors influence the angiogenesis of wound healing, including the microenvironment in which vascular morphogenesis occurs. Soluble polypeptides, cell-cell and cell-matrix interactions, and hemodynamic and biomechanical forces all play strategic roles. More recently, we have learned that blood vessel sprouting during wound healing is likely to be critically dependent on a well-ordered signaling cascade responsible for regulating microvascular cytoskeletal function. In addition, clear-cut roles for the extracellular matrix and the repertoire of metalloproteinases controlling matrix remodeling also play modulatory roles in fostering wound-healing angiogenesis.
Investigations and procedures for examining and measuring healing, migration, and formation of new blood vessels that occur during the response to injury, and which are impaired during chronic wound healing during diabetes, venous stasis ulceration, pressure ulcer formation and ischemia-reperfusion disorders: all, are known in the art. See, for example, Demidova-Rice et al., Lasers in Surgery and Medicine 39:706-715 (2007); Kutcher et al., Am. J. Pathol. 171:693-701 (2007); Herman, et al., Stewart Martin, ed. (2007); Herman, D. Shepro, ed. Elsevier Pub, Inc. (2006); Riley et al., J. Burns & Wounds 4:141-59, (2005); Papetti et al., Am. J. Physiol 282:947-970, (2002); Papetti et al., Am. J. Pathol. 159:165-77 (2001); the disclosures of which are all incorporated herein by reference.
There is, therefore, a great interest and need in developing compositions and devices that are useful for treating wounds. See, for example, Gandy, U.S. Patent Publication No. 20080213238; Gandy U.S. Patent Publication No. 20060142198; Gandy U.S. Patent Publication No. 20060004189; Gandy U.S. Patent Publication No. 20050191286; and Gandy U.S. Patent Publication No. 20040197319, the disclosures of which are all incorporated herein by reference.
Other attempts to prepare wound healing compositions are disclosed in, for example, Knighton, U.S. Pat. Nos. 5,165,938 and 4,957,742, which disclose platelet enriched plasma produced from blood wherein the platelets are activated by thrombin which causes the release of platelet-derived growth and angiogenesis factors. A carrier, such as a microcrystalline collagen, is added to produce a wound-treating salve, and the composition is applied directly to wounds and initiates healing in non-healing wounds as well as accelerating normal wound-healing by increasing vascularization, stimulating fibroblast mitosis and migration, and increasing collagen synthesis by fibroblasts. It is said that the composition may also be applied to tissue to facilitate the growth of hair.
Worden, U.S. Pat. No. 6,524,568, discloses a platelet gel wound healing composition that includes growth factors and ascorbic acid and optionally including an antioxidant such as Vitamin A and/or Vitamin E. Antibiotics may also be included. Chao, U.S. Pat. No. 5,185,160, discloses a heat-treated, viral-inactivated platelet membrane microparticle fraction which may be prepared from outdated platelets. The microparticle fraction is said to be substantially free of platelet ghosts and may be used as a pharmaceutical preparation in transfusions. Chao, U.S. Pat. No. 5,332,578, also discloses a heat-treated, viral-inactivated platelet membrane microparticle product which may be prepared from outdated mammalian platelets. The microparticle product is said to contain isolated platelet membrane fragments that are free of alloantigens and GP IIb/IIIa complexes and it is said that the product may be used as a pharmaceutical preparation in transfusions.
Crowe, U.S. Patent Publication No. U.S. 2004/0265293A1, discloses a dehydrated composition that includes freeze-dried platelets. The platelets are loaded with trehalose in an amount from about 10 mM to about 50 mM, and at a temperature of from greater than about 25° C. to less than about 40° C. The freeze-dried platelets are said to be substantially shelf-stable and are rehydratable so as to have a normal response to an agonist, for example, thrombin, and it is said that virtually all of the platelets participate in clot formation within about three minutes at 37° C.
Van der Meulen et al., J. Membrane Biol. 71:47-59 (1983), discloses porcine alpha-granules that were found to be essentially homogeneous by transmission electron microscopy. Freeze-fractured samples of isolated granules revealed intramembranous particles on the exoplasmic fracture surface and, to a lesser extent, on the protoplasmic fracture surface, whereas the PS (protoplasmic) surface was relatively smooth and, it is said, the granules appeared to be sealed. Membranes were isolated by alkali extraction of the granules which removed protein and phospholipids yielding membrane vesicles devoid of the dense core. The membranes were said to contain major and minor polypeptides. The exposure of specific proteins on the cytoplasmic surface of the granule membrane was also determined. In sealed granules, bands were modified by the reagents, and a fraction eluted by alkali extraction was also analyzed and found to contain nine major polypeptides.
Chao et al., Transfusion 36:536-542 (1996), discloses preparation of IPM from outdated platelets. The platelets were disrupted by freezing and thawing, washed and heated to inactivate possible viral contaminants, and then a sonicated membrane microvesicle fraction was separated and lyophilized. The hemostatic activity of IPM was measured by its ability to reduce the prolonged bleeding time in thrombocytopenic rabbits. According to Chao, administration of IPM at a dose of 2 mg per kg results in a substantial reduction in the bleeding time. It is reported that, in a series of 23 experiments, a median preinjection bleeding time of 15 minutes was reduced to 6 minutes within 4 hours after IPM administration. Administration of IPM was said to show a mild enhancement in the thrombogenicity index, as measured in the Wessler rabbit model, which was not significant. Chao concludes that IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.
Gogstad, Thrombosis Research 20:669-681 (1980), discloses a method for the isolation of alpha-granules wherein a two-step French pressure cell homogenization procedure produced an organelle concentrate for loading on density gradients. The procedure was said to be optimalized with respect to recovery of intact alpha-granules. The organelle homogenate was loaded to 17.5-27.5% metrizamide gradients and centrifuged. Organelle aggregate formation was said to be minimized by controlling the ionic conditions and the shape of the gradient. The alpha-granules were separated from lysosomes and dense bodies, but overlapped with the mitochondria, and the alpha-granules were recovered from the gradient to omit the major amount of mitochondria from the final preparation.
Hernandez, Vox Sang 73:36-42 (1997), discloses an investigation into the effects on hemostatsis of nonliving platelet derivatives. The effects of different platelet preparations on primary hemostatsis in a well-established perfusion model were evaluated, and studies were carried out with blood anticoagulated with low molecular weight heparin. Frozen-thawed, sonicated or lyophilized platelets were added to normal blood or to blood which had been experimentally depleted of platelets. Platelet interaction with the subendothelium and fibrin deposition were morphometrically evaluated. Hernandez reports that addition of non-viable platelet preparations to thrombocytopenic blood promoted a statistically significant increase in the deposition of fibrin on the subendothelium, but only lyophilized platelets retained some ability to interact with the subendothelium. Flow cytometry studies demonstrated the presence of GPIb, GPIIa and P-selection on lyophilized platelets. Hernandez concludes that preparations containing non-viable platelets may still retain some hemostatic properties.
Blood vessels are the method by which oxygen and nutrients are circulated and supplied to tissue, as well as the method by which waste products are removed from such tissue. Angiogenesis refers to the process by which new blood vessels are formed from preexisting blood vessels. See, for example, the review by Folkman and Shing, J. Biol. Chem. 267:10931-10934 (1992), Dvorak et al., J. Exp. Med. 174:1275-1278 (1991). Accordingly, angiogenesis is generally considered an essential biological process, which includes instances where a greater degree of angiogenesis is desired, such as wound healing, as discussed above. However, abnormal or inappropriate angiogenesis, where there is excessive blood vessel proliferation, can lead to severe negative outcomes, as exemplified in vascularized ocular diseases such as proliferative diabetic retinopathy, and wet age-related macular degeneration and in cancers, where solid tumor growth has been demonstrated to be angiogenesis-dependent, with the newly developed or angiogenic microvessels transforming dormant avascular micrometastases into actively growing macroscopic tumors, which derive an ample supply of oxygen and nutrients from angiogenic tumor microvessels.
It is known that tumor growth impacts a large number of people each year, e.g., Cancer accounts for 7.1 million deaths annually (12.5% of the global total). Approximately 20 million people suffer from cancer; a figure projected to rise to 30 million within 20 years. The number of new cases annually is estimated to rise from 10 million to 15 million by 2020 (World Health Organization).
With respect to angiogenesis-dependent visually blinding disorders accompanying aging and diabetes, there is a comparably staggering and ever-increasing number of affected individuals. For example, with respect to visually blinding disorders observed during aging, angiogenic or vascular complications of AMD account for roughly 10 percent of all those patients suffering with AMD but, “wet” AMD accounts for 90 percent of all AMD-associated blindness: roughly 2.3 million of the 34 million Americans over age 70 will be affected. And, with respect to the visually blinding vascular complications associated with diabetes, it is likely that between 40 to 45 percent of Americans diagnosed with diabetes have some stage of diabetic retinopathy (www.nei.nih.gov).
Cancer is becoming an increasingly important factor in the global burden of disease. The estimated number of new cases annually is expected to rise from 10 million in 2000 to 15 million by 2020. Some 60% of these cases will occur in the less developed parts of the world. More than 7 million people now die each year from cancer. Yet with the existing knowledge, at least one-third of cancer cases that occur annually throughout the world could be prevented. The use of therapies designed to inhibit angiogenesis or neovascularization may significantly effect the growth of solid tumors and development of ocular disease. Through blocking angiogensis or neovascularization, tumor growth and ocular disease can be inhibited suggesting that these diseases require the continued blood vessel growth for progression of the tumors or ocular disease. Inhibition of angiogenesis or neovascularization is, therefore, a promising anti-cancer treatment and an anti-ocular angiogenic therapeutic approach as has recently been demonstrated (anti-VEGF).
Inhibition of angiogenesis may also be useful in treating diseases that are characterized by unregulated blood vessel development including, for example, vascular tumors (e.g., sarcomas, carcinomas, and lymphomas) and ocular diseases (e.g., macular degeneration and diabetic retinopathy). Cancer cells, as used herein, include tumors, tissue, and the like.
As can be seen from the above, there is a great need for and interest in developing compounds and compositions that are useful in treating and healing wounds, such as chronic wounds caused by diabetes which are difficult to heal. Not all current methods of treating chronic wounds have been successful. It is therefore an object of the present disclosure to identify new compounds and methods that will promote and improve wound healing, especially for chronic and otherwise unhealable wounds. Compounds and compositions that specifically promote keratinocyte migration to a wound, and which may not promote substantial keratinocyte proliferation, on the one hand, and which enhance endothelial cell formation, and which may not promote substantial endothelial cell migration or proliferation, would represent a significant advance over the products that are currently available for healing wounds. Further, there is great need for and interest in developing compounds and compositions that are useful in combating vascular tumors and certain vision-threatening complications resulting from abnormal angiogenesis, such as, for example, diabetic retinopathy or age-related macular degeneration. It is therefore another object of the present disclosure to identify novel compounds and compositions that inhibit abnormal angiogenesis, but do not ablate the complement of physiologic blood vessels or survival of normal cells and tissues that are required for normal organismic functionality. Disclosure of such approaches and/or entities would represent a significant advance over the current state of the art, standard of care or products that are currently available or are in use.